Matrix Assisted Laser Desorption Ionisation (“MALDI”) is a method of generating ions of analyte substances. It is a particularly successful technique for the generation of ions of large organic and biochemical molecules for which many other ionisation techniques are largely unsuccessful. The analyte material is dissolved in an appropriate solvent. A droplet of the solution and a droplet of another solution of an appropriate matrix material are then placed on the surface of a sample or target plate such that the two solutions are allowed to mix. The resulting solution is then allowed to evaporate and the residual matrix material and analyte material form small crystals. The sample or target plate is then placed in a mass spectrometer and the sample or target plate is irradiated with a pulsed laser. The crystals are normally irradiated with ultra violet (UV) light, although infra red (IR) light may be used with certain matrix materials.
Since the ions are generated using a pulsed laser beam, the resulting ions are produced in short pulses. A particularly convenient type of mass spectrometer for analysing ions generated from a pulsed ion source is a Time of Flight (“TOF”) mass spectrometer.
Linear Time of Flight mass analysers are known wherein pulses of ions are accelerated with a high voltage, typically between 10 kV and 30 kV. The time the ions take to pass through a flight tube and arrive at an ion detector is recorded. Since the ions are all accelerated to approximately the same kinetic energy then the resulting velocities of the ions will be inversely proportional to the square root of their mass, assuming that the ions are all singly charged. Accordingly, the time for ions to reach the ion detector is also proportional to the square root of their mass.
In a MALDI ion source ions may be desorbed from the surface of a sample or target plate with a range of velocities. The mean velocity of the desorbed ions has been determined to be approximately independent of the mass to charge ratio of the ions and is typically between 300–600 m/s. The actual mean velocity of the desorbed ions will depend upon the laser power used and the size and nature of the sample and matrix crystals. It has been observed that the desorbed ions tend to have a considerable range of velocities about the mean velocity. As a consequence, the ions accelerated into a Time of Flight mass spectrometer will normally have a wide range of ion energies which can create problems when using a Time of Flight mass analyser.
In a linear Time of Flight mass spectrometer the arrival time of ions at the ion detector is dependent upon the energy of the ions. Accordingly, if the ions released from an ion source have a range of kinetic energies then they will also have a range of arrival times. This gives rise to broad mass peaks and poor mass resolution.
It is known to attempt to address this problem by using a reflectron wherein ions are reflected through nearly 180° and pass back through a portion of the flight tube to the ion detector. Ions that have relatively higher initial kinetic energies prior to entering the reflectron will therefore penetrate further into the reflectron before being reflected. Ions having relatively higher kinetic energies will therefore have a further overall distance to travel. In this way ions which are initially faster and more energetic can be made to travel a greater distance before striking the ion detector. If the mean flight path in the reflectron is arranged appropriately, then to a first approximation ions can be arranged to arrive at the ion detector substantially independent of the kinetic energy which they possess upon arriving at the acceleration region of the Time of Flight mass analyser. Using a reflectron therefore results in narrower observed mass peaks and an improved mass resolution. A MALDI ion source coupled to a Time of Flight mass analyser incorporating a reflectron is therefore able to achieve a higher mass resolution than a MALDI ion source coupled to a linear Time of Flight mass analyser without a reflectron.
A MALDI Time of Flight mass analyser incorporating a reflectron is also able to separate and analyse fragment ions resulting from parent ions which spontaneously decompose during flight. Such parent ions are generally metastable ions and the process of decomposition in flight is commonly referred to as Post Source Decay (“PSD”). The decomposition of parent ions may also be induced by collision with gas molecules in, for example, a fragmentation or collision cell. Such a process is commonly referred to as Collision Induced Decomposition (“CID”).
Fragment ions which are produced in a field free flight region can be considered to retain, to a first approximation, essentially the same velocity as their corresponding parent ions (although in reality the velocity of the fragment ions may be very slightly increased or decreased as a result of energy released during the decomposition reaction). Therefore, to a first approximation, the fragment ions will arrive at the ion detector of a linear Time of Flight mass spectrometer which does not have a reflectron at substantially the same time as any corresponding unfragmented parent ions. Parent ions and corresponding fragment ions are not therefore substantially temporally separated using a linear Time of Flight mass analyser which does not have a reflectron. If a mass spectrometer incorporating a reflectron is used then the situation is different. Since a fragment ion has approximately the same velocity as its corresponding parent ion, but has a lower mass, then it follows that the fragment ion must have a lower kinetic energy than that of its corresponding parent ion. For example, if a parent ion has a mass to charge ratio of 2000 and the parent ion fragments into a fragment ion having a mass to charge ratio of 1000, then the fragment ion will possess only half the kinetic energy which the parent ion originally had. The ratio of the kinetic energies of the fragment and parent ions will be in the same ratio as their masses. Since the fragment ion will have a lower kinetic energy than its corresponding parent ion, the fragment ion will penetrate to a shallower depth into the reflectron and will therefore follow a shorter overall path. Consequently, if fragment ions are formed either by CID or by PSD in a mass spectrometer incorporating a reflectron then such fragment ions will arrive at the ion detector before any corresponding related unfragmented parent ions. If the reflectron is optimised to reflect lower energy fragment ions then more energetic parent ions will not be reflected by the reflectron and hence such parent ions may become lost to the system. Therefore, it is possible to separate fragment ions from any corresponding unfragmented parent ions using an appropriately arranged Time of Flight mass analyser incorporating a reflectron and to separately record and mass analyse the fragment ions.
The analysis of fragment ions is particularly useful for determining the structure and identity of corresponding parent ions. For bio-polymer ions it may be possible to deduce their molecular sequence from fragment ion and parent ion data.
In order to analyse PSD fragment ions a Time of Flight mass analyser incorporating a reflectron may be used. In a linear field reflectron the optimal energy focusing at the ion detector is achieved when the time of flight within the reflectron is approximately equal to the overall time of flight in the field free region upstream and downstream of the reflectron. The time of flight of fragment ions in the reflectron region depends upon the depth of penetration of the fragment ions into the reflectron. For relatively low energy fragment ions the depth of penetration into the reflectron may be increased such that the depth of penetration of the ions is closer to the optimum. This can be achieved by stepping down the reflectron voltage. The reflectron voltage may, for example, be stepped through a number of voltage settings. A 25% reduction in reflectron voltage from step to step may be used to progressively focus fragment ions having lower mass to charge ratios and hence lower kinetic energies. Selected data (or segments of individual mass spectra) relating to ions focussed by the reflectron from each step may then be merged or stitched together to form a single or composite mass spectrum relating to all the various fragment ions produced from the fragmentation of a particular parent ion.
A known MALDI Time of Flight mass spectrometer used to analyse fragment ions comprises a timed electrostatic deflecting system or ion gate situated in a flight tube upstream of the Time of Flight mass analyser. The ion gate is arranged such as to allow only ions having a specific velocity to pass therethrough. The timing of the ion gate is such that only parent ions having a small range of mass to charge ratios will be transmitted by the ion gate. Any fragment ions produced by fragmentation of parent ions upstream of the ion gate will also travel at essentially the same velocity as the corresponding unfragmented parent ions. Accordingly, such fragment ions will also be transmitted by the ion gate at substantially the same time as related unfragmented parent ions. Therefore, the use of the ion gate allows the recording of fragment ions originating from just one particular parent ion (or from a smaller number of parent ions).
The known mass spectrometer suffers from a number of problems associated with the use of a timed ion gate to select particular ions. Timed ion gates have the disadvantage that they can perturb the motion of the ions of interest i.e. those ions intended to be transmitted by the ion gate. Transmitted ions can also be axially and/or radially accelerated or decelerated by stray electric fields from the ion gate. The fast electronic pulse required to gate the ions may also be too slow or may overshoot and oscillate. This adversely affects both the parent ion and the fragment ion mass resolution and the overall ion transmission of the mass spectrometer. Low energy fragment ions are particularly vulnerable to the affects of stray electric fields from the ion gate.
A known ion gate as used in a conventional mass spectrometer comprises a Bradbury Nielson ion gate. A Bradbury Nielson ion gate comprises parallel wires with voltages of alternating polarity applied to successive wires to minimise stray fields. Such an arrangement suffers from the problem that the parallel wires may reduce ion transmission since some ions will strike the wires and become neutralised.
Other effects resulting from the use of ion gates can also be detrimental. For example, ions that are deliberately deflected by an ion gate can strike other parts of the mass spectrometer and may produce scattered ions (or other secondary particles) by sputtering, secondary ion emission, surface induced decomposition or similar processes. As a result, the observation of less intense fragment ions from less intense parent ions in complex mixtures may be obscured by the presence of scattered or secondary ions caused by the deliberate deflection of more abundant ions when the ion gate is closed.
Another problem with using a timed ion gate is that it only allows a fragment ion spectrum for one particular parent ion to be recorded at any one time. Therefore, in order, for example, to characterise a complex mixture of peptide ions by PSD it is necessary to set the ion gate to transmit each individual parent peptide ion in the mixture in turn and to separately record the corresponding fragment ion spectrum for each parent ion by stepping down the voltage applied to the reflectron. It can therefore take a considerable amount of time to obtain fragment ion spectra for all the parent ions. Furthermore, the conventional approach can consume relatively small samples before all parent peptide ions have been analysed. This problem is also further compounded by the fact that not all parent peptide ions will yield useful fragment ions by PSD. However, it will not be known which parent peptide ions will yield the most useful data until after all parent ions been individually analysed. As a result, a lot of time and sample may be consumed acquiring PSD fragment ion data from less productive or relating to less interesting parent peptide ions. In some cases all of the sample may be consumed before any useful or interesting data has been acquired.
On the other hand, if a timed ion gate is not incorporated into a conventional mass analyser then all the fragment ions resulting from fragmentation of all the numerous parent ions will be transmitted and recorded at the same time. Accordingly, if the sample being analysed comprises a complex mixture of different parent peptide ions then the resulting mass spectrum will be impossible to analyse since the mass spectrum will be completely swamped with mass peaks and it will not be known which of very numerous observed fragment ions correspond with which parent ions. As a consequence, it will not be possible to relate observed fragment ions to particular parent ions and hence no useful information can be obtained if a conventional mass spectrometer is used without an ion gate.
It is therefore desired to provide an improved mass spectrometer and method of mass spectrometry.